Sequencing and analysis of thermophylic Streptomyces plasmid pTSC2 and characterization of its rolling-circle replication
高温链霉菌质粒pTSC2的测序分析和滚环复制方式的鉴定

Objective] A Streptomyces strain X3 -3, growing up to 50 ℃, was isolated from the composts prepared from swine manure and straw. This strain harbored a -7-kb plasmid pTSC2. Objective ] Cloning, sequencing and analyzing pTSC2, and identifying its replication mode as well. Methods ] The complete nucleotide sequence of pTSC2 was obtained by sub-cloning and primer-walking ; the replication gene (rep) , double-strand origin (dso) and single-strand origin (sso) were identified by multiple sequence alignments; and the replication intermediate was detected by Southern hybridization after neutral transfer. Results] The complete nucleotide sequence of pTSC2 consisted of 7516-bp in length, encoded eight proteins, four of which resembled the replication and conjugation proteins of Streptomyces plasmid pIJ101 which replicates in a rolling-circle mode. The replication elements, rep, dso and sso, were also similar with pIJ101. The rep and dso was proved as essential components for plasmid replication by transformation of both S. lividans ZX7 and thermophilic Streptomyces spp. 2C, while sso was dispensable for replication. Southern hybridization detected a large amount of single-stranded DNA accumulated during replication. Conclusion ] We identified that the thermophilic Streptomyces plasimd pTSC2 replicated in a rolling-circle mode. It was first reported that the plasmid from thermophilic Streptomyces was cloned and sequenced, as well as the replication of which was characterized